Tumor hypoxia has been shown in several clinical studies to be a prognostic indicator of poor treatment outcome. These findings have been independent of therapeutic modality, with hypoxie tumors that are treated surgically doing equally as poorly as those treated with radiation therapy. Genetic models in mice, and patients with VHL-deficient tumors, have suggested that the ability to induce genes in response to hypoxia with the transcription factor HIF-1 may play a stimulatory role in tumorigenesis. Hypoxia, however, is also a potent repressor of gene expression, but few studies have investigated the mechanism of hypoxia-dependent gene downregulation. We present the hypothesis that gene repression by hypoxia can contribute to tmnorigenesis, and the NC2 (negative co-factor 2) molecules are responsible for at least some of this repression. NC2alpha/beta (Drl/DRAP) was originally characterized as a biochemical activity that was able to repress transcription in vitro. We have preliminary evidence that repressive activity is induced in hypoxic cells, and extracts from hypoxic cells fail to transcribe (at least) some templates in vitro. Biochemical characterization of this activity suggests that NC2 may be a general repressor of transcription in hypoxia. NC2 induction by hypoxia could then contribute to tumor formation through the downregulation of such hypoxia-repressed target genes as tumor suppressors thrombospondin 1 and 2 (TSP1/TSP2), apoptotic inhibitors stathmin and survivin, and tumor marker alpha fetal protein (AFP). We therefore propose to establish a genetic system for the analysis of NC2's role in tumor development. We will address this goal with three specific aims, first establish that NC2 is responsible for hypoxic repression of specific target promoters in vivo, second perform structure-function relationship of NC2alpha and NC2beta to identify domains that are critical for hypoxic gene repression, and third construct tumor cell lines with compromised NC2 activity that test proof in principle that hypoxic gene repression is important in model tumor formation. These studies will determine if hypoxic gene repression through NC2 activity could be a potential therapeutic target for future molecular therapeutics in the treatment of human cancers.